Retroviral vectors are relevant for a range of applications, including gene therapy. However, progress in lentiviral gene therapy, for example, has been hampered by the requirement for production of purified lentiviral vectors with high titre.
Lentiviral vectors are typically generated by a packaging cell which releases vector particles into the supernatant. Since lentiviral vectors are labile, subsequent purification methods must use physiological (or non-harsh) conditions as much as possible to maximize recovery of the vector. Further, the methodology needs to be scalable and cost-effective.
Currently, lentiviral particles are usually purified from supernatant by ultracentrifugation. This is a laborious process, which only provides a 40% viral recovery and cannot be easily scaled. Other methods for the purification have been explored, for example ultrafiltration—which provides a 50% recovery using a 750 kda membrane, low density gradient centrifugation or anion exchange chromatography. All of these methods are cumbersome and laborious and relatively unproven. Additionally, these methods result in the concentration of envelope proteins as well as other cellular components that hinder the infectivity of the viral titre.
Affinity chromatography may be used as a single-step capture method for the generic recovery of viral vectors by exploiting streptavidin and biotin interactions. Nesbeth et al. (Molecular Therapy 2006, 13, 814-822) engineered a novel human 293T based packaging cell line BL15, which metabolically produces spontaneously biotin-tagged lentiviral vectors requiring only biotin in the culture medium. This metabolic biotinylation technology facilitates highly efficient affinity-mediated paramagnetic-particle and chromatographic capture of viral particles.
A similar system has been described for adenovirus (Parrott et al. (2003) Mol. Ther. 8:688-700), in which the fiber capsid protein is genetically fused to a biotin acceptor peptide, which is metabolically biotinylated during vector production by the endogenous biotin ligase in 293 cells.
However, the value of such biotinylation systems for purification of viral vectors in manufacturing is limited for two main reasons: since the affinity of biotin to streptavidin is very high, subsequent removal of the virus from the streptavidin matrix is difficult and requires harsh conditions. Further, since these methods require presence of biotin, residual free biotin competes with the streptavidin matrix for binding (Nesbeth et al. 2006 (as above); Williams et al. (2005) Biotechnology and Bioengineering 89: 783-787; and Williams et al. (2005) Journal of Chromatography B 820: 111-119).
There have also been various reports of strategies to aid purification of recombinant viral vectors by engineering the viral envelope protein to include some kind of tag.
WO2007/095201 describes a viral vector comprising a recombinant viral envelope protein consisting of a rhabdovirus viral envelope, such as VSV-G, engineered with a heterologous polypeptide. The heterologous polypeptide is cloned between the SU and TM unit of the envelope. Peptide-tagged-viral particles are subsequently purified by metal ion affinity chromatography.
WO2014/121005 describes a viral vector comprising an epitope-tagged viral envelope whereby the epitopes, CD118, V5 or HA, are cloned after the signal peptide or after the proline rich region (PRR) of viral envelope glycoproteins. Subsequent purification of the supernatant relies on a centrifugation upon harvesting of epitope-tagged viral particles followed by incubation with antibodies against the three epitopes. Purified particles are then eluted by adding the antigen ie the epitope of the antibodies.
Ye et al (2004, J. Virol. 78:9820-9827) engineered a metal binding peptide-tagged MLV envelope by incorporating the peptide into a part of hypervariable region of the viral protein. Subsequent viral purification then involved immobilized metal affinity chromatography.
WO2004/000220 describes tagging the spike protein of VSV-G by insertion of a His-6 peptide tag. Virus then may be isolated a purified by column affinity chromatography or sedimentation with magnetic beads.
A disadvantage of such systems is that insertion of a tagging protein into the reading frame of a viral envelop protein can disrupt the functional integrity of the envelope protein and negatively impact viral titer.
This issue is illustrated by studies aimed at genetically engineering the viral envelope glycoprotein for cell-specific viral transduction. The viral envelope glycoprotein of Moloney leukemia virus (MLV) is the most commonly altered envelope for targeted transduction with modifications including: peptide insertion in pre-folded domains; expression of peptides as additional domains; and peptides fused directly to the transmembrane part of the envelope.
Even though some of these studies generated correctly folded chimeric envelopes that were be able to bind its specific receptor on target cells, most N-terminally substituted chimeric envelopes studied to date have either had very low viral incorporation or absence of transduction of target cells. For example, gammaretroviral vectors with envelope proteins modified to the stromal cell derived factor 1-alpha (Katane et al., 2002 EMBO Rep. 3, 899-904. doi:10.1093/embo-reports/kvf179) or an integrin binding peptide (Wu et al., 2010 Cancer Res. 70, 9549-9553. doi:10.1158/0008-5472.CAN-10-1760) were shown to have poor transduction efficiencies.
The genetic engineering of viral envelope proteins such as MLV, VSV-G and RD114 therefore remains a technical challenge for the field. This is due to the delicate interaction between the binding and fusion domains. Their dependent activities, when altered by peptide insertions, seem to inhibit infection and in turn negatively impact viral titer. This is true for methods involving altering envelope glycoproteins in order to tag vector for purification as well as methods involving altering envelope glycoproteins in order to enable cell-specific viral transduction.
Thus there is a need for methods for producing and purifying retroviral vectors which are not associated with these disadvantages.